Superagonists and antagonists of interleukin-2

ABSTRACT

Novel human interleukin-2 (IL-2) muteins or variants thereof, and nucleic acid molecules and variants thereof are provided. Methods for producing these muteins as well as methods for stimulating the immune system of an animal are also disclosed. In addition, the invention provides recombinant expression vectors comprising the nucleic acid molecules of this invention and host cells into which expression vectors have been introduced. Pharmaceutical compositions are included comprising a therapeutically effective amount of a human IL-2 mutein of the invention and a pharmaceutically acceptable carrier. The IL-2 muteins can be used in pharmaceutical compositions for use in treatment of cancer and in stimulating the immune response.

This invention was made with U.S. Government support under Grant no.AI51321 awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

This application is a Continuation of U.S. patent application Ser. No.16/219,786, filed Dec. 13, 2018, which is a Division of U.S. patentapplication Ser. No. 15/217,416, filed Jul. 22, 2016, now U.S. Pat. No.10,183,980, which is a Continuation of U.S. patent application Ser. No.13/997,503, filed Oct. 10, 2013, now U.S. Pat. No. 9,428,567, which isbased on International Application No. PCT/US2011/066911, filed Dec. 22,2011, which claims the benefit of U.S. Provisional Application No.61/426,307, filed Dec. 22, 2010, now expired, the disclosures of whichare incorporated herein by reference in their entirety for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM,LISTING APPENDIX SUBMITTED ON A COMPACT DISK

This invention incorporated by reference the Sequence Listing text copysubmitted herewith, which was created on May 31, 2018, entitled068597_5019 US ST25.txt which is 28 kilobytes in size.

BACKGROUND

Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily byactivated CD4⁺T cells, which plays a crucial role in producing a normalimmune response. IL-2 promotes proliferation and expansion of activatedT lymphocytes, potentiates B cell growth, and activates monocytes andnatural killer cells. It was by virtue of these activities that IL-2 wastested and is used as an approved treatment of cancer (aldesleukin,Proleukin®).

In eukaryotic cells human IL-2 is synthesized as a precursor polypeptideof 153 amino acids, from which 20 amino acids are removed to generatemature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has beenproduced in E. coli (Rosenberg 1984), in insect cells (Smith 1985) andin mammalian COS cells (Taniguchi 1983).

IL-2 works by interacting with three different receptors: theinterleukin 2 receptor alpha (IL-2Rα; CD25), the interleukin 2 receptorbeta (IL-2Rβ;CD122), and the interleukin 2 receptor gamma (IL-2Rγ;CD132;common gamma chain). The first receptor to be identified was the IL-2Rα,which is a 55 kD polypeptide (p55) that appears upon T cell activationand was originally called Tac (for T activation) antigen. The IL-2Rαbinds IL-2 with a K_(d) of approximately 10⁻⁸ M, and is also known asthe “low affinity” IL-2 receptor. Binding of IL-2 to cells expressingonly the IL-2Rα does not lead to any detectable biologic response.

The IL-2Rβ is a member of the type I cytokine receptor familycharacterized by the two cysteine/WSXWS motif. The IL-2Rβ is expressedcoordinately with the IL-2Rγ. The IL-2Rγ, a 64 kD polypeptide, is alsoknown as the common γ chain because it is shared among a number ofcytokine receptors, including the receptor for interleukin-4 andinterleukin-7. The IL-2Rβγ is the same signaling receptor complex thatcan bind to IL-15.

Most cells, for example, resting T cells are insensitive to IL-2 sincethey only express the IL-2Rβ and the IL-2Rγ. Upon antigenreceptor-mediated T cell activation, the IL-2Rαis rapidly expressed.Once the IL-2Rα binds IL-2, it then sequentially engages the IL-2Rβ andthe IL-2Rγ (FIG. 1). IL-2 binding by the IL-2Rαβγ complex results insignal transduction through a Jak/STAT signaling pathway and IL-2mediated growth stimulation.

So far, only limited structure/function analysis of human IL-2 hasoccurred, although analysis of mouse IL-2 has been extensive (Zurawski,S. M. and Zurawski, (1989) Embo J 8: 2583-90; Zurawski, S. M, et. al.,(1990) Embo J 9: 3899-905; Zurawski, G. (1991). Trends Biotechnol 9:250-7; Zurawski, S. M. and Zurawski, G. (1992) Embo J 11: 3905-10.Zurawski, et. al., EMBO J, 12 5113-5119 (1993)). Some human IL-2 muteinshave been examined for their activity on human PHA blasts (Xu, et. al.,Eur. Cytokine Netw, 6, 237-244 (1995)). Other examples of human IL-2muteins are provided by Buchli and Ciardelli, Arch. Biochem. Biophys,307(2): 411-415, (1993), Collins, L., et al., PNAS USA 85:7709-7713(1988), and U.S. Pat. No. 5,696,234 (Zurawski et al.).

The use of IL-2 as an antineoplastic agent has been limited by theserious toxicities that accompany the doses necessary for a tumorresponse. The major side effect of IL-2 therapy is vascular leaksyndrome (VLS), which leads to the accumulation of intravascular fluidin the lungs and liver resulting in pulmonary edema and liver damage.Until recently it was believed that VLS was caused by the release ofproinflammatory cytokines from IL-2 activated NK cells. However, arecent report points to the direct binding of IL-2 to lung endothelialcells, as a purported cause of VLS. (Krieg et al.,PNAS USA107(26)11906-11911 (2010). In principle, an IL-2 variant with highaffinity for IL-2Rβ, whose activity was not dependent on CD25 expressioncould have improved clinical utility and reduced toxicity.

One IL-2 mutein of clinical interest is BAY 50-4798, which differs fromwild-type IL-2 by the substitution of arginine for asparagine atposition 88 (R88N) (Steppan et al. (2006) J. Interferon and CytokineRes., 26(3): 171-. This modification allegedly results in an IL-2 muteinwith relatively reduced binding to the IL-2Rf3γ, and thought to possesslower toxicity relative to wild type IL-2. However, a clinical studyfound that patients receiving BAY 50-4798 experienced a similar degreeof IL-2 mediated VLS.

For these reasons, it is clear that IL-2 muteins that exhibit uniqueproperties are needed. Potential uses of such muteins include treatingcancer (as a direct and/or adjunct therapy) and immunodeficiency (e.g.,HIV and tuberculosis). Other potential uses of IL-2 are derived from itsimmunostimulatory activity, and include direct treatment of cancer,treating immunodeficiency, such as HIV or human SCID patients; treatinginfectious disease, such as tuberculosis; its use as an adjuvant in“cancer vaccine” strategies; and for immune system stimulationindications, such as enhancing standard vaccination protocols (e.g.,elderly). For example, IL-2 muteins that exhibit reduced VLS would beadvantageous.

The present disclosure provides novel IL-2 muteins.

SUMMARY OF THE INVENTION

IL-2 exerts a wide spectrum of effects on the immune system, and itplays crucial roles in regulating both immune activation andhomeostasis. As an immune system stimulator, IL-2 has found use in thetreatment of cancer and chronic viral infections. The stimulatoryaffects of IL-2 can also cause havoc, mediating autoimmunity andtransplant rejection. Because of its instrumental role in immuneregulation and disease, the identification of IL-2 molecules withimproved qualities remains an active area of research.

To these ends, the instant disclosure provides novel IL-2 compositionsbased on new insights into how IL-2 interacts with its cognatereceptors. In most circumstances, IL-2 works through three differentreceptors: the IL-2Rα, the IL-2Rβ, and the IL-2Rγ. Most cells, such asresting T cells, are not responsive to IL-2 since they only express theIL-2Rβ, and the IL-2Rγ, which have low affinity for IL-2. Uponstimulation, resting T cells express the relatively high affinity IL-2receptor IL-2Rα. Binding of IL-2 to the IL-2Rαcauses this receptor tosequentially engage the IL-2Rβ, and the IL-2Rγ, bringing about T cellactivation.

Based on a structural analysis of the interaction of IL-2 with itsreceptors, mutant forms of IL-2 were made which possess relativelyincreased affinity for the IL-2 Rβ when compared to wild-type IL-2, suchthat IL-2 mediated stimulation no longer requires engagement of theIL-2Rα. Such mutants are potent IL-2 signaling agonists.

Thus, in one embodiment an IL-2Rβ binding protein is disclosed, whereinthe equilibrium dissociation constant for the IL-2Rβ is less than thatof wild-type human IL-2.

Using these novel “super”-agonist IL-2 molecules as a starting point,“super”-antagonists were made which can bind IL-2Rβ, reducing theinteraction of IL-2Rβ with IL-2Rγ and receptor signaling.

In other embodiments, a method for producing an IL-2Rβ binding proteinis described, the method encompassing mutating a human IL-2, producing afirst generation IL-2 mutein, identifying a first generation IL-2 muteinwith an equilibrium dissociation constant for an interleukin-2 receptorβ less than that of wild-type human IL-2, mutating the identified firstgeneration IL-2 mutein, producing a second generation IL-2 mutein,identifying a second generation IL-2 mutein which binds and/or signalsthrough an interleukin-2 receptor γ relatively less than wild-type IL2,thereby generating an IL-2 binding protein.

Collectively, these IL-2 muteins are referred to as “super-2s.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the interaction of IL-2 with itsreceptors and affects on T cells. The binding of IL-2 by the IL-2Rαresults in the sequential engagement of the IL-2Rβ and the IL-2Rγ. IL-2causes T cell clonal expansion and differentiation.

FIG. 2: FACS profile of IL-2 library. Products of error prone PCR of thehuman IL-2 gene were subjected to selection. The first generation IL-2library was generated through six rounds of selection. The first roundwas performed using tetrameric IL-2Rβ coupled to phycoerythrin (PE) tobind yeast expressing IL-2 muteins (A). Subsequent rounds of selectionwere accomplished using monomeric IL-2 Rβ labeled with PE. (B) Resultsfrom the second generation IL-2 library.

FIG. 3: Depicts the amino acid residues altered in the IL-2 muteinsshown relative to the wild-type IL-2 sequence. The binding affinity ofeach mutein and IL-2 for the IL-2Rβ is also shown.

FIG. 4: Stimulatory Effects of IL-2 Muteins on CD25⁻ and CD25⁺ NaturalKiller Cells. Dose response relationships of wildtype IL-2 and the IL-2muteins 6-6, D10, and H9 on STAT5 phosphorylation witnessed in treated(A) CD25⁻ and (B) CD25⁺ YT-1 Natural Killer cells. Circles wild-typeIL-2; squares 6-6; triangles up H9; triangles down D10.

FIG. 5: CD25 Independence of IL-2 Mutein Binding. Dose response curvesof STAT5 phosphorylation for CD25⁻ and CD25⁺ YT-1 Natural Killer cells.(A) IL-2 and IL-2 (F42A) (circles, solid line wild-type IL-2, CD25+cells; squares, solid line IL-2 F42A, CD25+ cells; triangles up, dashedlines wild-type IL-2, CD25− cells; triangles down, dashed line, IL-2F42A, CD25− cells). (B) H9 and H9(F42A) (circles, solid line wild-typeH9, CD25+ cells; squares, solid line H9 F42A, CD25+ cells; triangles up,dashed lines H9, CD25− cells; triangles down, dashed line, H9 F42A,CD25− cells). While the F42A mutation right shifted the dose-responsecurve of wild-type IL-2 on CD25⁺ cells, but had no observable effect onCD25⁻, the dose response curves for H9 and H9 F42A were essentiallyoverlapping, regardless of CD25 expression.

FIG. 6: CD25 KO T cell stimulation. The ability of the disclosed IL-2muteins to stimulate T cells in the absence of the IL-2Rα was tested. Tcells isolated from CD25 knockout mice were stimulated with an IL-2mutein or wild-type IL-2. Dose response curves and respective EC50 ofIL-2 muteins are provided. As shown, all of the tested IL-2 muteinsresulted in relatively increased T cell stimulation, in the absence ofthe IL-2Ra, relative to wild-type IL-2.

FIG. 7: Human experienced CD4 T cell stimulation. FACS analysiscomparing the relative ability of the disclosed IL-2 muteins to induceexperienced T cell stimulation. T cells were stimulated with twoconcentrations (10 ng/ml or 1 ng/ml) of IL-2 mutein or wild-type IL-2.The percentage of stimulated T cells is shown in each FACS profile.

FIG. 8: Antibody dependent cellular cytotoxity (ADCC). The effect ofIL-2 mutein D10 on natural killer cell function, specificallyspontaneous and antibody-dependent cell mediated cytotoxicity wasassayed. Natural killer cells (effectors) and Cr⁵¹ labeled tumor cells(targets) were incubated together for 5 hours in the presence ofwild-type IL-2 or the IL-2 mutein D10, with or without the anti-EGFRantibody cetuximab. D10 stimulation of NK cell spontaneous cytotoxicitywas superior to high dose IL-2 (*p=0.008, **p=0.001) with minimalspontaneous cytotoxicity without IL-2 or D10 stimulation. Further,addition of D10 enhanced the ADCC of the cetuximab antibody.

FIG. 9: Crystal structure of D10. An initial hydrophobic core mutationof L85V led to a second generation IL-2 library targeting multiplehydrophobic core residues and a high affinity consensus sequence. Thecrystal structure of D10 contained well-defined electron density in theloop region preceding helix C.

FIG. 10: Novel IL-2 muteins exhibit enhanced stimulation of CD8⁺ T cellsbut not Tregs relative to IL-2. (A) Total cell counts of host CD3⁺ CD8⁺CD44^(high) memory-phenotype (MP) T cells and (B) host CD3⁺ CD4⁺CD25^(high) T cells (regulatory T cells) was determined in the spleensof mice receiving either PBS, 20 μg IL-2, 20 μg H9, or 1.5 μgIL-2/anti-IL-2 monoclonal antibody complexes (IL-2/mAb).

FIG. 11: Novel IL-2 muteins exhibit enhanced anti-tumor response withreduced adverse effects relative to IL-2. Pulmonary edema (pulmonary wetweight) served as a measure of adverse toxic effects following IL-2treatment, and was determined by weighing the lungs before and afterdrying (A). P values refer to comparisons between treatment modalities.*, p<0.05; **, p<0.01. (B) Anti-tumor properties of IL-2 muteins weretested in vivo using B16F10 melanoma cells. C57B1/6 mice (n=3-4mice/group) were injected subcutaneously with 106 B16F10 melanoma cellsfollowed by daily injections of either PBS, 20 μg IL-2, 20 μg H9, or 1.5μg IL- 2/anti-IL-2 monoclonal antibody complexes (IL-2/mAb) for fivedays once tumor nodules became visible and palpable, which typicallycorresponded to day 4 to 5 after tumor cell injections or a tumor sizeof about 15 mm². Shown is mean tumor area in mm²(+/−SD) vs. time upontumor inoculation. P values refer to comparison of IL-2 with the othertreatment modalities.

DETAILED DESCRIPTION

In order for the present disclosure to be more readily understood,certain terms and phrases are defined below as well as throughout thespecification.

Definitions

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley& Sons (New York, N.Y. 2001); and Sambrook and Russell, MolecularCloning: A Laboratory Manual 3rd ed., Cold Spring harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many terms used in the present disclosure. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

As used herein, “IL-2” means wild-type IL-2, whether native orrecombinant. Mature human IL-2 occurs as a 133 amino acid sequence (lessthe signal peptide, consisting of an additional 20 N-terminal aminoacids), as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983).The amino acid sequence of human IL-2 (SEQ ID NO: 1) is found in Genbankunder accession locator NP_000577.2. The amino acid sequence of maturehuman IL-2 is depicted in SEQ ID NO: 2. The murine (Mus musculus) IL-2amino acid sequence is found in Genbank under accession locator (SEQ IDNO: 3). The amino acid sequence of mature murine IL-2 is depicted in SEQID NO: 4.

SEQ ID NO: 1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS ITSTLT SEQ ID NO: 2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 3MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESA TVVDFLRRWIAFCQSIISTSPQSEQ ID NO: 4 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTS PQ

As used herein, “IL-2 mutein” means a polypeptide wherein specificsubstitutions to the interleukin-2 protein have been made. FIG. 3, forexample, discloses twelve IL-2 muteins and their corresponding relativebinding affinity for the IL-2Rβ. The IL-2 muteins can also becharacterized by amino acid insertions, deletions, substitutions andmodifications at one or more sites in or at the other residues of thenative IL-2 polypeptide chain. In accordance with this disclosure anysuch insertions, deletions, substitutions and modifications result in anIL-2 mutein that retains the IL-2Rβ binding activity. Exemplary muteinscan include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacids.

Muteins also include conservative modifications and substitutions atother positions of IL-2 (i.e., those that have a minimal effect on thesecondary or tertiary structure of the mutein). Such conservativesubstitutions include those described by Dayhoff in The Atlas of ProteinSequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785(1989). For example, amino acids belonging to one of the followinggroups represent conservative changes: Group I: ala, pro, gly, gln, asn,ser, thr; Group II: cys, ser, tyr, thr; Group III:val, ile, leu, met,ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; andGroup VI: asp, glu.

“Numbered in accordance with IL-2” means identifying a chosen amino acidwith reference to the position at which that amino acid normally occursin the mature sequence of wild type IL-2, for example R81 refers to theeighty-first amino acid, arginine, that occurs in SEQ ID NO: 2.

The term “cell types having the IL-2Rαβγ receptor” means the cells knownto have this receptor type, i.e., T cells, activated T cells, B cells,activated monocytes, and activated NK cells. The term “cell types havingthe IL-2Rβγ receptor” means the cells known to have that receptor type,i.e., B cells, resting monocytes, and resting NK cells.

The term “identity,” as used herein in reference to polypeptide or DNAsequences, refers to the subunit sequence identity between twomolecules. When a subunit position in both of the molecules is occupiedby the same monomeric subunit (i.e., the same amino acid residue ornucleotide), then the molecules are identical at that position. Thesimilarity between two amino acid or two nucleotide sequences is adirect function of the number of identical positions. In general, thesequences are aligned so that the highest order match is obtained. Ifnecessary, identity can be calculated using published techniques andwidely available computer programs, such as the GCS program package(Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN,FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequenceidentity can be measured using sequence analysis software such as theSequence Analysis Software Package of the Genetics Computer Group at theUniversity of Wisconsin Biotechnology Center (1710 University Avenue,Madison, Wis. 53705), with the default parameters thereof.

The term “polypeptide,” “protein” or “peptide” refer to any chain ofamino acid residues, regardless of its length or post-translationalmodification (e.g., glycosylation or phosphorylation).

In the event the mutant IL-2 polypeptides of the disclosure are“substantially pure,” they can be at least about 60% by weight (dryweight) the polypeptide of interest, for example, a polypeptidecontaining the mutant IL-2 amino acid sequence. For example, thepolypeptide can be at least about 75%, about 80%, about 85%, about90%,about 95% or about 99%, by weight, the polypeptide of interest.Purity can be measured by any appropriate standard method, for example,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

“Operably linked” is intended to mean that the nucleotide sequence ofinterest (i.e., a sequence encoding an IL-2 mutein) is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). “Regulatory sequences” include promoters, enhancers, and otherexpression control elements (e.g., polyadenylation signals). See, forexample, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionconstructs of the invention can be introduced into host cells to therebyproduce the human IL-2 muteins disclosed herein or to producebiologically active variants thereof

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but also to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell but are still included within the scope of the term as used herein.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, particle gun, orelectroporation.

As used herein, the term “pharmaceutically acceptable carrier” includes,but is not limited to, saline, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds (e.g., antibiotics) can also beincorporated into the compositions.

As used herein, we may use the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” to refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth). Hyperproliferativeand neoplastic disease states may be categorized as pathologic (i.e.,characterizing or constituting a disease state), or they may becategorized as non-pathologic (i.e., as a deviation from normal but notassociated with a disease state). The terms are meant to include alltypes of cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair. Theterms “cancer” or “neoplasm” are used to refer to malignancies of thevarious organ systems, including those affecting the lung, breast,thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, andthe genitourinary tract, as well as to adenocarcinomas which aregenerally considered to include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. An “adenocarcinoma” refersto a carcinoma derived from glandular tissue or in which the tumor cellsform recognizable glandular structures.

As used herein, the term “hematopoietic neoplastic disorders” includesdiseases involving hyperplastic/neoplastic cells of hematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. Preferably, the diseases arise from poorlydifferentiated acute leukemias (e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia). Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CIVIL)(reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97);lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Stemberg disease.

IL-2 Muteins

In various embodiments, the present disclosure provides IL-2polypeptides, which may be, but are not necessarily, substantiallypurified and that can function as an agonist of wild-type IL-2; carryingout one or more of the biological activities of IL-2 (e.g., stimulationof cellular proliferation)). IL-2 has been characterized as a T cellgrowth factor that induces proliferation of antigen-activated T cellsand stimulation of NK cells.

Also, described are IL-2 polypeptides that can function as an antagonistof wild-type IL-2; that is, preventing the biological activity of IL-2.

An exemplary mutant IL-2 polypeptide includes an amino acid sequencethat is at least about 80% identical to SEQ ID NO:2 which binds theIL-2R13 with an affinity that is greater than the affinity with whichthe polypeptide represented by SEQ ID NO: 2 binds the IL-2Rβ. Forexample, a mutant IL-2 polypeptide can have at least one mutation (e.g.,a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues)relative to a wild-type IL-2, and that binds the IL-2Rβ with higheraffinity than a wild-type IL-2.

An exemplary mutant IL-2 polypeptide can also include an amino acidsequence that is at least about 80% identical to SEQ ID NO: 2 and thatbinds to an IL-2 receptor γ (IL-2Rγ) with an affinity that is less thanthe affinity with which the polypeptide represented by SEQ ID NO: 2binds the IL-2Rγ. For example, a mutant IL-2 polypeptide can have atleast one mutation (e.g., a deletion, addition, or substitution of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or moreamino acid residues) relative to a wild-type IL-2, and that binds theIL-2Rγ with lower affinity than a wild-type IL-2.

Exemplary mutant IL-2 polypeptides can be at least about 50%, at leastabout 65%, at least about 70%, at least about 80%, at least about 85%,at least about 87%, at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% identical towild-type IL-2. The mutation can consist of a change in the number orcontent of amino acid residues. For example, the mutant IL-2 can have agreater or a lesser number of amino acid residues than wild-type IL-2.Alternatively, or in addition, an exemplary mutant polypeptide cancontain a substitution of one or more amino acid residues that arepresent in the wild-type IL-2. In various embodiments, the mutant IL-2polypeptide can differ from wild-type IL-2 by the addition, deletion, orsubstitution of a single amino acid residue, for example, a substitutionof the residue at position 69. Similarly, exemplary mutant polypeptidescan differ from wild-type by a substitution of two amino acid residues,for example, the residues at positions 24, 65, 74, 80, 81, 85, 86, 89,92, and 93 of SEQ ID NO:2.

By way of illustration, a polypeptide that includes an amino acidsequence that is at least 95% identical to a reference amino acidsequence of SEQ ID NO:2 is a polypeptide that includes a sequence thatis identical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid of SEQ ID NO: 2. Forexample, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence can occur at the amino (N—) or carboxy (C—)terminal positions of the reference amino acid sequence or anywherebetween those terminal positions, interspersed either individually amongresidues in the reference sequence or in one or more contiguous groupswithin the reference sequence.

The substituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. These mutations can be atamino acid residues that contact the IL-2Rβ and/or the IL-2Rγ.

More specifically, a mutation (whether conservative or non-conservative,by way of addition(s) or deletion(s)) can be made at one or more ofpositions. For example, the mutation can be: I24V, P65H, Q74R, Q74 H,Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I.The sequences of exemplary IL-2 muteins are as follows: 5-1 SEQ ID NO:5; 5-2 SEQ ID NO: 6; 6-6 SEQ ID NO: 7; A2 SEQ ID NO: 8; B1 SEQ ID NO: 9;B11 SEQ ID NO: 10; C5 SEQ ID NO: 11; D10 SEQ ID NO: 12; Eli) SEQ ID NO:13; G8 SEQ ID NO: 14; H4 SEQ ID NO: 15; and H9 SEQ ID NO:. 16.

SEQ ID NO: 5 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA R SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 6APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLARSKNFHLRPRD V ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 7APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLARSKNFHL I PRD V ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 8APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFHL T PRD VV SNINV FI LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 9APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SN V NV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 10 APTSSSTKKTQLQLEHLLLDLQM VLNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC LEEELKPLEEVLNLA S SKNFH FD PRD VVSNINV F VLELKGSETTFMCEYADETATIVEFL NRWITFCQSIISTLT SEQ ID NO: 11APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELK HLEEVLNLA N SKNFH VT PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 12APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA H SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 13APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRIVILTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 14APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA N SKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 15APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA S SKNFHL T PRD V ISNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 16APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH FD PRD VV SNINV F VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

With respect to affinity, this there are disclosed herein exemplarymutant IL-2 polypeptides that bind the IL-2Rβ with an affinity that ishigher than the wild type IL-2 polypeptide by at least about 2%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, or at least about 40% higher affinity or more. The wild-type IL-2polypeptide binds the IL-2Rβ with a K_(d) of about 280 nM. The bindingaffinity of exemplary disclosed mutant IL-2 polypeptides can also beexpressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 ormore fold higher affinity for the IL-2Rβ than wild-type IL-2.

Alternatively, or in addition, an exemplary mutant IL-2 polypeptide canhave increased potency in a T cell proliferation assay relative towild-type IL-2. The ability of a mutant IL-2 polypeptide to bind theIL-2Rβ can be assessed by numerous assays, including the cell bindingand proliferation assays described herein.

Exemplary mutant IL-2 polypeptides can have the ability to exhibit adecreased dissociation rate from the IL-2Rβ receptor subunit, such thatsignaling from the receptor/ligand complex persists for a longer timeperiod following transient exposure to the mutant IL-2 polypeptide, ascompared to a wild-type IL-2.

Also provided in the instant disclosure are mutant polypeptides thatdisrupt the association of the IL-2Rβ with the IL-2Rγ such that thisinteraction is reduced by about 2%, about 5%, about 10%, about 15%,about 20%, about 50%, about 75%, about 90%, about 95% or more relativeto wild-type IL-2.

Exemplary mutant IL-2 polypeptides possessing both properties ofincreased affinity for the IL-2Rβ and disruption of the IL-2Rβ with theIL-2Rγ interaction are also disclosed.

As described further below, an exemplary class of mutant IL-2polypeptides is provided in the instant disclosure with increasedbinding affinity for the IL-2Rβ and/or decreased binding affinity to theIL-2Rγ using yeast surface display relative to wild-type IL-2.

Structural Comparison of IL-2 Muteins with Wild-type IL-2 RecombinantExpression of IL-2 Muteins, Expression Vectors and Host Cells

In various embodiments, polypeptides used in the practice of the instantinvention are synthetic, or are produced by expression of a recombinantnucleic acid molecule. In the event the polypeptide is a chimera (e.g.,a fusion protein containing at least a mutant IL-2 polypeptide and aheterologous polypeptide), it can be encoded by a hybrid nucleic acidmolecule containing one sequence that encodes all or part of the mutantIL-2, and a second sequence that encodes all or part of the heterologouspolypeptide. For example, the mutant IL-2 polypeptide may be fused to ahexa-histidine tag to facilitate purification of bacterially expressedprotein, or to a hemagglutinin tag to facilitate purification of proteinexpressed in eukaryotic cells.

Methods for constructing a DNA sequence encoding the IL-2 muteins andexpressing those sequences in a suitably transformed host include, butare not limited to, using a PCR-assisted mutagenesis technique.Mutations that consist of deletions or additions of amino acid residuesto an IL-2 polypeptide can also be made with standard recombinanttechniques. In the event of a deletion or addition, the nucleic acidmolecule encoding IL-2 is optionally digested with an appropriaterestriction endonuclease. The resulting fragment can either be expresseddirectly or manipulated further by, for example, ligating it to a secondfragment. The ligation may be facilitated if the two ends of the nucleicacid molecules contain complementary nucleotides that overlap oneanother, but blunt-ended fragments can also be ligated. PCR-generatednucleic acids can also be used to generate various mutant sequences.

The complete amino acid sequence can be used to construct aback-translated gene. A DNA oligomer containing a nucleotide sequencecoding for IL-2 mutein can be synthesized. For example, several smalloligonucleotides coding for portions of the desired polypeptide can besynthesized and then ligated. The individual oligonucleotides typicallycontain 5′ or 3′ overhangs for complementary assembly.

In addition to generating mutant polypeptides via expression of nucleicacid molecules that have been altered by recombinant molecularbiological techniques, mutant polypeptides can be chemicallysynthesized. Chemically synthesized polypeptides are routinely generatedby those of skill in the art.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the DNA sequences encoding an IL-2 mutein will be inserted intoan expression vector and operatively linked to an expression controlsequence appropriate for expression of the IL-2 mutein in the desiredtransformed host. Proper assembly can be confirmed by nucleotidesequencing, restriction mapping, and expression of a biologically activepolypeptide in a suitable host. As is well known in the art, in order toobtain high expression levels of a transfected gene in a host, the genemust be operatively linked to transcriptional and translationalexpression control sequences that are functional in the chosenexpression host.

The DNA sequence encoding the IL-2 mutein, whether prepared by sitedirected mutagenesis, chemical synthesis or other methods, can alsoinclude DNA sequences that encode a signal sequence. Such signalsequence, if present, should be one recognized by the cell chosen forexpression of the IL-2 mutein. It can be prokaryotic, eukaryotic or acombination of the two. It can also be the signal sequence of nativeIL-2. The inclusion of a signal sequence depends on whether it isdesired to secrete the IL-2 mutein from the recombinant cells in whichit is made. If the chosen cells are prokaryotic, it generally ispreferred that the DNA sequence not encode a signal sequence. If thechosen cells are eukaryotic, it generally is preferred that a signalsequence be encoded and most preferably that the wild-type IL-2 signalsequence be used.

IL-2 Mutein Fusion Proteins

As noted above, exemplary mutant IL-2 polypeptides can be prepared asfusion or chimeric polypeptides that include a mutant IL-2 polypeptideand a heterologous polypeptide (i.e., a polypeptide that is not IL-2 ora mutant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplaryheterologous polypeptides can increase the circulating half-life of thechimeric polypeptide in vivo, and may, therefore, further enhance theproperties of the mutant IL-2 polypeptides. In various embodiments, thepolypeptide that increases the circulating half-life may be a serumalbumin, such as human serum albumin, or the Fc region of the IgGsubclass of antibodies that lacks the IgG heavy chain variable region.Exemplary Fc regions can include a mutation that inhibits complementfixation and Fc receptor binding, or it may be lytic, i.e., able to bindcomplement or to lyse cells via another mechanism, such asantibody-dependent complement lysis (ADCC; USSN 08/355,502 filed Dec.12, 1994).

The “Fc region” can be a naturally occurring or synthetic polypeptidethat is homologous to the IgG C-terminal domain produced by digestion ofIgG with papain. IgG Fc has a molecular weight of approximately 50 kDa.The mutant IL-2 polypeptides can include the entire Fc region, or asmaller portion that retains the ability to extend the circulatinghalf-life of a chimeric polypeptide of which it is a part. In addition,full-length or fragmented Fc regions can be variants of the wild-typemolecule. That is, they can contain mutations that may or may not affectthe function of the polypeptides; as described further below, nativeactivity is not necessary or desired in all cases.

The Fc region can be “lytic” or “non-lytic,” but is typically non-lytic.A non-lytic Fc region typically lacks a high affinity Fc receptorbinding site and a C′1q binding site. The high affinity Fc receptorbinding site of murine IgG Fc includes the Leu residue at position 235of IgG Fc. Thus, the Fc receptor binding site can be destroyed bymutating or deleting Leu 235. For example, substitution of Glu for Leu235 inhibits the ability of the Fc region to bind the high affinity Fcreceptor. The murine C′1q binding site can be functionally destroyed bymutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG.For example, substitution of Ala residues for Glu 318, Lys 320, and Lys322 renders IgG1 Fc unable to direct antibody-dependent complementlysis. In contrast, a lytic IgG Fc region has a high affinity Fcreceptor binding site and a C′1q binding site. The high affinity Fcreceptor binding site includes the Leu residue at position 235 of IgGFc, and the C′1q binding site includes the Glu 318, Lys 320, and Lys 322residues of IgG1. Lytic IgG Fc has wild-type residues or conservativeamino acid substitutions at these sites. Lytic IgG Fc can target cellsfor antibody dependent cellular cytotoxicity or complement directedcytolysis (CDC). Appropriate mutations for human IgG are also known(see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; andBrekke et al., The Immunologist 2: 125, 1994).

In other embodiments, the chimeric polypeptide can include the mutantIL-2 polypeptide and a polypeptide that functions as an antigenic tag,such as a FLAG sequence. FLAG sequences are recognized by biotinylated,highly specific, anti-FLAG antibodies, as described herein (see alsoBlanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad.Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptidefurther comprises a C-terminal c-myc epitope tag.

In other embodiments, the chimeric polypeptide includes the mutant IL-2polypeptide and a heterologous polypeptide that functions to enhanceexpression or direct cellular localization of the mutant IL-2polypeptide, such as the Aga2p agglutinin subunit (see, e.g., Boder andWittrup, Nature Biotechnol. 15:553-7, 1997).

In other embodiments, a chimeric polypeptide including a mutant IL-2 andan antibody or antigen-binding portion thereof can be generated. Theantibody or antigen-binding component of the chimeric protein can serveas a targeting moiety. For example, it can be used to localize thechimeric protein to a particular subset of cells or target molecule.Methods of generating cytokine-antibody chimeric polypeptides aredescribed, for example, in U.S. Pat. No. 6,617,135.

Nucleic Acid Molecules Encoding Mutant IL-2

In some embodiments the mutant IL-2 polypeptide, either alone or as apart of a chimeric polypeptide, such as those described above, can beobtained by expression of a nucleic acid molecule. Just as mutant IL-2polypeptides can be described in terms of their identity with wild-typeIL-2 polypeptides, the nucleic acid molecules encoding them willnecessarily have a certain identity with those that encode wild-typeIL-2. For example, the nucleic acid molecule encoding a mutant IL-2polypeptide can be at least 50%, at least 65%, preferably at least 75%,more preferably at least 85%, and most preferably at least 95% (e.g.,99%) identical to the nucleic acid encoding wild-type IL-2 (e.g., SEQ IDNO:2). The nucleic acid sequence encoding mature IL-2 and its signalsequence are found in SEQ ID NO: 17.

The nucleic acid molecules provided can contain naturally occurringsequences, or sequences that differ from those that occur naturally,but, due to the degeneracy of the genetic code, encode the samepolypeptide. These nucleic acid molecules can consist of RNA or DNA (forexample, genomic DNA, cDNA, or synthetic DNA, such as that produced byphosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of IL-2)can also be included. Those of ordinary skill in the art of molecularbiology are familiar with routine procedures for isolating nucleic acidmolecules. They can, for example, be generated by treatment of genomicDNA with restriction endonucleases, or by performance of the polymerasechain reaction (PCR). In the event the nucleic acid molecule is aribonucleic acid (RNA), molecules can be produced, for example, by invitro transcription.

Exemplary isolated nucleic acid molecules of the present disclosure caninclude fragments not found as such in the natural state. Thus, thisdisclosure encompasses recombinant molecules, such as those in which anucleic acid sequence (for example, a sequence encoding a mutant IL-2)is incorporated into a vector (e.g., a plasmid or viral vector) or intothe genome of a heterologous cell (or the genome of a homologous cell,at a position other than the natural chromosomal location).

As described above, the mutant IL-2 polypeptide of the invention mayexist as a part of a chimeric polypeptide. In addition to, or in placeof, the heterologous polypeptides described above, a nucleic acidmolecule of the invention can contain sequences encoding a “marker” or“reporter.” Examples of marker or reporter genes include β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)), dihydrofolatereductase (DHFR), hygromycin-B-hosphotransferase (HPH), thymidine kinase(TK), lacz (encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter.

The nucleic acid molecules of the invention can be obtained byintroducing a mutation into IL-2-encoding DNA obtained from anybiological cell, such as the cell of a mammal. Thus, the nucleic acidsof the invention (and the polypeptides they encode) can be those of amouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon,dog, or cat. In one embodiment, the nucleic acid molecules will be thoseof a human.

Expression of Mutant IL-2 Gene Products

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto mutant IL-2 polypeptides, expression vectors containing a nucleicacid molecule encoding a mutant IL-2 polypeptide and cells transfectedwith these vectors are among the preferred embodiments.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences described herein. Neither will all hosts function equally wellwith the same expression system. However, one of skill in the art maymake a selection among these vectors, expression control sequences andhosts without undue experimentation. For example, in selecting a vector,the host must be considered because the vector must replicate in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. For example, vectors thatcan be used include those that allow the DNA encoding the IL-2 muteinsto be amplified in copy number. Such amplifiable vectors are well knownin the art. They include, for example, vectors able to be amplified byDHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufmanand Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA Gene:Analysis of Signals Utilized for Efficient Expression”, Mol. Cell.Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase (“GS”)amplification (see, e.g., U.S. Pat. No. 5,122,464 and European publishedapplication 338,841).

In some embodiments, the human IL-2 muteins of the present disclosurewill be expressed from vectors, preferably expression vectors. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of coding sequences to which they are operablylinked. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids (vectors). However, otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses, and adeno-associated viruses) areincluded also.

Exemplary recombinant expression vectors can include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, operably linked to the nucleic acid sequence to beexpressed.

The expression constructs or vectors can be designed for expression ofan IL-2 mutein or variant thereof in prokaryotic or eukaryotic hostcells.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and other standard molecularbiology laboratory manuals.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters. Strategies to maximize recombinant protein expression in E.coli can be found, for example, in Gottesman (1990) in Gene ExpressionTechnology: Methods in Enzymology 185 (Academic Press, San Diego,Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res.20:2111-2118. Processes for growing, harvesting, disrupting, orextracting the IL-2 mutein or variant thereof from cells aresubstantially described in, for example, U.S. Pat. Nos. 4,604,377;4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798;4,748,234; and 4,931,543, herein incorporated by reference in theirentireties.

In some embodiments the recombinant IL-2 muteins or biologically activevariants thereof can also be made in eukaryotes, such as yeast or humancells. Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cerenvisiae include pYepSecl (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2^(nd) ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.).

The sequences encoding the human IL-2 muteins of the present disclosurecan be optimized for expression in the host cell of interest. The G-Ccontent of the sequence can be adjusted to levels average for a givencellular host, as calculated by reference to known genes expressed inthe host cell. Methods for codon optimization are well known in the art.Codons within the IL-2 mutein coding sequence can be optimized toenhance expression in the host cell, such that about 1%, about 5%, about10%, about 25%, about 50%, about 75%, or up to 100% of the codons withinthe coding sequence have been optimized for expression in a particularhost cell.

Vectors suitable for use include T7-based vectors for use in bacteria(see, for example, Rosenberg et al., Gene 56:125, 1987), the pMSXNDexpression vector for use in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988), and baculovirus-derived vectors (for example, theexpression vector pBacPAK9 from Clontech, Palo Alto, Calif.) for use ininsect cells.

In some embodiments nucleic acid inserts, which encode the polypeptideof interest in such vectors, can be operably linked to a promoter, whichis selected based on, for example, the cell type in which expression issought.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the actualDNA sequence encoding the IL-2 mutein of this invention, particularly asregards potential secondary structures. Hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the DNA sequences of thisinvention, their secretion characteristics, their ability to fold thepolypeptides correctly, their fermentation or culture requirements, andthe ease of purification of the products coded for by the DNA sequences.

Within these parameters one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe desired DNA sequences on fermentation or in large scale animalculture, for example, using CHO cells or COS 7 cells.

The choice of expression control sequence and expression vector, in someembodiments, will depend upon the choice of host. A wide variety ofexpression host/vector combinations can be employed. Useful expressionvectors for eukaryotic hosts, include, for example, vectors withexpression control sequences from SV40, bovine papilloma virus,adenovirus and cytomegalovirus. Useful expression vectors for bacterialhosts include known bacterial plasmids, such as plasmids from E. coli,including col E1, pCRI, pER32z, pMB9 and their derivatives, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages. Useful expression vectors foryeast cells include the 2 μ plasmid and derivatives thereof. Usefulvectors for insect cells include pVL 941 and pFastBac™ 1 (GibcoBRL,Gaithersburg, Md.). Cate et al., “Isolation Of The Bovine And HumanGenes For Mullerian Inhibiting Substance And Expression Of The HumanGene In Animal Cells”, Cell, 45, pp. 685-98 (1986).

In addition, any of a wide variety of expression control sequences canbe used in these vectors. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol sequences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the trp system, the TAC or TRCsystem, the major operator and promoter regions of phage lambda, forexample PL, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., PhoA, the promoters of the yeast a-matingsystem, the polyhedron promoter of Baculovirus, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells or their viruses, and various combinations thereof

A T7 promoter can be used in bacteria, a polyhedrin promoter can be usedin insect cells, and a cytomegalovirus or metallothionein promoter canbe used in mammalian cells. Also, in the case of higher eukaryotes,tissue-specific and cell type-specific promoters are widely available.These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body.Skilled artisans are well aware of numerous promoters and otherregulatory elements which can be used to direct expression of nucleicacids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neo^(r)) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that can be used in the invention include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes a mutant IL-2 polypeptide are also features of theinvention. A cell of the invention is a transfected cell, i.e., a cellinto which a nucleic acid molecule, for example a nucleic acid moleculeencoding a mutant IL-2 polypeptide, has been introduced by means ofrecombinant DNA techniques. The progeny of such a cell are alsoconsidered within the scope of the invention.

The precise components of the expression system are not critical. Forexample, a mutant IL-2 polypeptide can be produced in a prokaryotichost, such as the bacterium E. coli, or in a eukaryotic host, such as aninsect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells,NIH 3T3 cells, or HeLa cells). These cells are available from manysources, including the American Type Culture Collection (Manassas, Va.).In selecting an expression system, it matters only that the componentsare compatible with one another. Artisans or ordinary skill are able tomake such a determination. Furthermore, if guidance is required inselecting an expression system, skilled artisans may consult Ausubel etal. (Current Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A LaboratoryManual, 1985 Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

In some embodiments, IL-2 muteins obtained will be glycosylated orunglycosylated depending on the host organism used to produce themutein. If bacteria are chosen as the host then the IL-2 mutein producedwill be unglycosylated. Eukaryotic cells, on the other hand, willglycosylate the IL-2 muteins, although perhaps not in the same way asnative-IL-2 is glycosylated. The IL-2 mutein produced by the transformedhost can be purified according to any suitable method. Various methodsare known for purifying IL-2. See, e.g. Current Protocols in ProteinScience, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg,David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, JohnWiley and Sons, Inc. IL-2 muteins can be isolated from inclusion bodiesgenerated in E. coli, or from conditioned medium from either mammalianor yeast cultures producing a given mutein using cation exchange, gelfiltration, and or reverse phase liquid chromatography.

Another exemplary method of constructing a DNA sequence encoding theIL-2 muteins is by chemical synthesis. This includes direct synthesis ofa peptide by chemical means of the protein sequence encoding for an IL-2mutein exhibiting the properties described. This method can incorporateboth natural and unnatural amino acids at positions that affect theinteractions of IL-2 with the IL-2Rα, the IL-2Rβ and/or the IL-2Rγ.Alternatively a gene which encodes the desired IL-2 mutein can besynthesized by chemical means using an oligonucleotide synthesizer. Sucholigonucleotides are designed based on the amino acid sequence of thedesired IL-2 mutein, and preferably selecting those codons that arefavored in the host cell in which the recombinant mutein will beproduced. In this regard, it is well recognized that the genetic code isdegenerate—that an amino acid may be coded for by more than one codon.For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) iscoded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W)is coded for by a single codon, TGG. Accordingly, it will be appreciatedthat for a given DNA sequence encoding a particular IL-2 mutein, therewill be many DNA degenerate sequences that will code for that IL-2mutein. For example, it will be appreciated that in addition to thepreferred DNA sequence for mutein 5-2 shown in FIG. 2, there will bemany degenerate DNA sequences that code for the IL-2 mutein shown. Thesedegenerate DNA sequences are considered within the scope of thisdisclosure. Therefore, “degenerate variants thereof” in the context ofthis invention means all DNA sequences that code for and thereby enableexpression of a particular mutein.

The biological activity of the IL-2 muteins can be assayed by anysuitable method known in the art. Such assays include PHA-blastproliferation and NK cell proliferation.

Methods of Treatment

In some embodiments, mutant IL-2 polypeptides, and/or nucleic acidsexpressing them, can be administered to a subject to treat a disorderassociated with abnormal apoptosis or a differentiative process (e.g.,cellular proliferative disorders or cellular differentiative disorders,such as cancer, by, for example, producing an active or passiveimmunity). In the treatment of such diseases, the disclosed IL-2 muteinsmay possess advantageous properties, such as reduced vascular leaksyndrome.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver. Thecompositions of the present invention (e.g., mutant IL-2 polypeptidesand/or the nucleic acid molecules that encode them) can also beadministered to a patient who has a viral infection (e.g., AIDS or aninfluenza).

The mutant IL-2 polypeptides can be used to treat patients who have, whoare suspected of having, or who may be at high risk for developing anytype of cancer, including renal carcinoma or melanoma, or any viraldisease. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The termalso includes carcinosarcomas, which include malignant tumors composedof carcinomatous and sarcomatous tissues.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders.

Other examples of proliferative and/or differentiative disorders includeskin disorders. The skin disorder may involve the aberrant activity of acell or a group of cells or layers in the dermal, epidermal, orhypodermal layer, or an abnormality in the dermal-epidermal junction.For example, the skin disorder may involve aberrant activity ofkeratinocytes (e.g., hyperproliferative basal and immediately suprabasalkeratinocytes), melanocytes, Langerhans cells, Merkel cells, immunecell, and other cells found in one or more of the epidermal layers,e.g., the stratum basale (stratum germinativum), stratum spinosum,stratum granulosum, stratum lucidum or stratum corneum. In otherembodiments, the disorder may involve aberrant activity of a dermalcell, for example, a dermal endothelial, fibroblast, immune cell (e.g.,mast cell or macrophage) found in a dermal layer, for example, thepapillary layer or the reticular layer.

Examples of skin disorders include psoriasis, psoriatic arthritis,dermatitis (eczema), for example, exfoliative dermatitis or atopicdermatitis, pityriasis rubra pilaris, pityriasis rosacea, parapsoriasis,pityriasis lichenoiders, lichen planus, lichen nitidus, ichthyosiformdermatosis, keratodermas, dermatosis, alopecia areata, pyodermagangrenosum, vitiligo, pemphigoid (e.g., ocular cicatricial pemphigoidor bullous pemphigoid), urticaria, prokeratosis, rheumatoid arthritisthat involves hyperproliferation and inflammation of epithelial-relatedcells lining the joint capsule; dermatitises such as seborrheicdermatitis and solar dermatitis; keratoses such as seborrheic keratosis,senile keratosis, actinic keratosis, photo-induced keratosis, andkeratosis follicularis; acne vulgaris; keloids and prophylaxis againstkeloid formation; nevi; warts including verruca, condyloma or condylomaacuminatum, and human papilloma viral (HPV) infections such as venerealwarts; leukoplakia; lichen planus; and keratitis. The skin disorder canbe dermatitis, e.g., atopic dermatitis or allergic dermatitis, orpsoriasis.

Patients amenable to treatment may also have psoriasis. The term“psoriasis” is intended to have its medical meaning, namely, a diseasewhich afflicts primarily the skin and produces raised, thickened,scaling, nonscarring lesions. The lesions are usually sharply demarcatederythematous papules covered with overlapping shiny scales. The scalesare typically silvery or slightly opalescent. Involvement of the nailsfrequently occurs resulting in pitting, separation of the nail,thickening and discoloration. Psoriasis is sometimes associated witharthritis, and it may be crippling. Hyperproliferation of keratinocytesis a key feature of psoriatic epidermal hyperplasia along with epidermalinflammation and reduced differentiation of keratinocytes. Multiplemechanisms have been invoked to explain the keratinocytehyperproliferation that characterizes psoriasis. Disordered cellularimmunity has also been implicated in the pathogenesis of psoriasis.Examples of psoriatic disorders include chronic stationary psoriasis,psoriasis vulgaris, eruptive (gluttate) psoriasis, psoriaticerythroderma, generalized pustular psoriasis (Von Zumbusch), annularpustular psoriasis, and localized pustular psoriasis.

Alternatively, or in addition to methods of direct administration topatients, in some embodiments, mutant IL-2 polypeptides can be used inex vivo methods. For example, cells (e.g., peripheral blood lymphocytesor purified populations of lymhocytes isolated from a patient and placedor maintained in culture) can be cultured in vitro in culture medium andthe contacting step can be affected by adding the IL-2 mutant to theculture medium. The culture step can include further steps in which thecells are stimulated or treated with other agents, e.g., to stimulateproliferation, or to expand a population of cells that is reactive to anantigen of interest (e.g., a cancer antigen or a viral antigen). Thecells are then administered to the patient after they have been treated.

Pharmaceutical Compositions and Methods of Administration

In some embodiments, mutant IL-2 polypeptides and nucleic acids can beincorporated into compositions, including pharmaceutical compositions.Such compositions typically include the polypeptide or nucleic acidmolecule and a pharmaceutically acceptable carrier.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. The mutant IL-2 polypeptides of theinvention may be given orally, but it is more likely that they will beadministered through a parenteral route. Examples of parenteral routesof administration include, for example, intravenous, intradermal,subcutaneous, transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral applicationcan include the following components: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfate; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such asmono- and/or di-basic sodium phosphate, hydrochloric acid or sodiumhydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants,e.g., sodium dodecyl sulfate. Prevention of the action of microorganismscan be achieved by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof

Oral compositions, if used, generally include an inert diluent or anedible carrier. For the purpose of oral therapeutic administration, theactive compound can be incorporated with excipients and used in the formof tablets, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel™, or corn starch; a lubricant such as magnesium stearate orSterotes™; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In the event of administration by inhalation, the mutant IL-2polypeptides, or the nucleic acids encoding them, are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of the mutant IL-2 polypeptides or nucleic acidscan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

In some embodiments, compounds (mutant IL-2 polypeptides or nucleicacids) can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In some embodiments, compounds (mutant IL-2 polypeptides or nucleicacids) can also be administered by transfection or infection usingmethods known in the art, including but not limited to the methodsdescribed in McCaffrey et al. (Nature 418:6893, 2002), Xia et al.(Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst.Pharm. 53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325,1996).

In one embodiment, the mutant IL-2 polypeptides or nucleic acids areprepared with carriers that will protect the mutant IL-2 polypeptidesagainst rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing standard techniques. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Dosage, toxicity and therapeutic efficacy of such mutant IL-2polypeptides or nucleic acids compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED_(50.)Compounds that exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a mutant IL-2polypeptides (i.e., an effective dosage) depends on the polypeptideselected. For instance, single dose amounts in the range ofapproximately 0.001 to 0.1 mg/kg of patient body weight can beadministered; in some embodiments, about 0.005, 0.01, 0.05 mg/kg may beadministered. In some embodiments, 600,000 IU/kg is administered (IU canbe determined by a lymphocyte proliferation bioassay and is expressed inInternational Units (IU) as established by the World Health Organization1^(st) International Standard for Interleukin-2 (human)). The dosage maybe similar to, but is expected to be less than, that prescribed forPROLEUKIN®. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of the mutant IL-2 polypeptides of theinvention can include a single treatment or, can include a series oftreatments. In one embodiment, the compositions are administered every 8hours for five days, followed by a rest period of 2 to 14 days, e.g., 9days, followed by an additional five days of administration every 8hours.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

EXAMPLES

The invention is further described in, and illustrated by, the followingstudies that serve as non-limiting examples.

Example 1: Functional Expression of IL-2 on the Surface of Yeast

Although IL-2 has been displayed on bacteriophage previously (Buchli etal., Arch. Biochem. Biophys. 339:79-84, 1997), the prior system was notamenable to directed evolution and therefore not suitable for obtainingIL-2 mutants with improved binding for subunits of the IL-2R. Toovercome this, IL-2 was expressed on the surface of yeast cells. HumanIL-2 DNA was cloned into yeast display vector pCT302. Saccharomycescerevisiae strain EBY100 was transformed with the pCT302 IL-2 vector andgrown for 3 days at 30° C. on SD-CAA plates. Individual colonies of IL-2yeast were grown overnight at 30° C. in SD-CAA, then introduced in SGCAAfor 2 days at 20° C. The yeast were stained with tetramerizedbiotinylated IL-2Rβ, biotinylated γ or biotinylated IL-2Rβ in thepresence of biotinylated y. The ectodomains of IL-2Rβ and γ wereC-terminally biotinylated and coupled to phycoerythrin-conjugaedstrepavidin for use as a staining and sorting reagent. IL-2 tetramerswere formed by incubating 2 μM of biotinylated IL-2Rβ with 470 nMstreptavidin-phycoerythrin (SA-PE, Invitrogen) for 15 minutes on ice.These receptor “tetramers” enhanced the avidity of the low affinitymonomeric ectodomain (ECD) interactions with IL-2, enabling maximalrecovery of IL-2 variants from libraries. Similar to solution wild-typeIL-2, yeast-displayed IL-2 bound weakly to IL- 2Rβ alone, did not bindto at all to γ alone, but did bind to γ in the presence of IL-2Rβ, asevidenced by diagonal staining seen by flow cytometry (data not shown).Thus, the yeast-displayed IL-2 recapitulates the cooperative assembly ofthe heterodimeric receptor complex on cells seen with soluble IL-2, andis therefore suitable as a platform for library selection.

Example 2: Construction and Screening of an IL-2 Mutant Library

The first generation in vitro strategy was to create an error-prone PCRlibrary of the entire IL-2 gene. The first generation mutant IL-2library was constructed as follows. Wildtype human interleukin-2 (IL-2)was subjected to error-prone mutagenesis using the GeneMorph® II RandomMutagenesis kit following the manufacturer's instructions. The followingprimers were used for error-prone PCR: 5′-GCACCTACTTCAAGTTCTAC-3′(“IL-2_errprone_for) and 5′-GCCACCAGAGGATCC-3′ (”IL-2_errprone_rev). Theproduct of the error prone PCR reaction was then amplified using thefollowing primers:5′AGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGCACCTACTTCAAGTTCTAC-3′ and5′ACACTGTTGTTATCAGATCTCGAGCAAGTCTTCTTCGGAGATAAGCTTTTGTTCGCCACCAGAGGATCC-3′ to yield approximately 130 μg of DNA. Yeast displayvector pCT302 was double digested with restriction enzymes Nhel andBamHI and gel purified. The IL-2 DNA and the pCT302 DNA were mixedtogether in a 5:1 μg ratio with electrocompetent EBY100 yeast. The yeastwere electroporated to facilitate entry of the library DNA into theyeast. This electroporation was repeated approximately 20 times to yielda final library size of 1×10⁸transformants.

Selection of first generation IL-2 library: The library was subjected tosix rounds of selection against IL-2Rβ (FIG. 2A). In the first round,the library was labeled with 470 nM tetrameric IL-2Rβ, which was formedby mixing 2 μM biotinylated IL-2Rβ with 470 nMstreptavidin-phycoerythrin conjugate (SAV-PE) for 15 min. The librarywas incubated with IL-2Rβ for 1.5 h, washed with PBS-BSA buffer(phosphate buffered saline+bovine serum albumin), and incubated withMiltenyi anti-PE MicroBeads for 20 min at 4° C. The cells were againwashed and flowed over a magnetic column for selection. This selectionmethod was successively repeated five more times with alterations onlyin IL-2Rβ concentration (round 2-1 μM, round 3 -1 round 4-300 nM, round5-300 nM, round 6-100 nM, all monomeric IL-2Rβ). Upon conclusion ofselections, round five and round six yeast cultures were spread onSD-CAA plates, which yielded individual yeast colonies. Eighteenresulting yeast colonies were tested for binding to 500 nM IL-2Rβ. TheIL-2 DNA isolated from these eighteen yeast colonies was sequenced.Amino acid differences among these eighteen yeast colonies relative tothe corresponding residue in wildtype IL-2 is shown in Table 1.

TABLE 1 residue # 5 34 43 61 74 75 77 81 85 103 106 112 120 wt IL-2 S PK E Q S N R L F E A R 5_1 R 5_2 V 5_3 V D 5_4 K Y 5_5 V 5_6 R S 5_8 (wt)5_9 R V 5_10 V 6_1 V 6_2 R R V 6_3 N V 6_4 V 6_5 V 6_6 I V 6_7 I V 6_8 KV 6_10 T V

Library construction of second generation IL-2 library: Based on thehigh percentage of clones containing L85V, a second IL-2 library wasconstructed that focused primarily on hydrophobic core residues. Asite-directed IL-2 library was constructed with mutations at Q74, L80,R81, L85, 186, 189, 192, V93. Q74 was allowed to vary as H/K/N/Q/R/S.R81 was allowed to vary at all 20 amino acids with the NNK degeneratecodon, where N represents a 25% mix each of adenine, thymine, guanine,and cytosine nucleotides and K is either guanine or thymine. Theremaining residues were allowed to vary as F/I/L/V. The library wasconstructed by assembly PCR using the following oligos:

IL-2_affmat_ass01 GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCAIL-2_affmat_ass02 CAAAATCATCTGTAAATCCAGAAGTAAATGCTCCAGTTGTAGCTGTGIL-2_affmat_ass03 GGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAIL-2_affmat_ass04B AACTTAGCTGTGAGCATCCTGGTGAGTTTGGGATTCTTGTAATTATTIL-2_affmat_ass05B GGATGCTCACAGCTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGIL-2_affmat_ass06 GTTCTTCTTCTAGACACTGAAGATGTTTCAGTTCTGTGGCCTTCTTGIL-2_affmat_ass07 CAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAIL-2_affmat_ass08 GTGAAAGTTTTTGCT SYK AGCTAAATTTAGCACTTCCTCCIL-2_affmat_ass09 AGCAAAAACTTTCAC NTCNNK CCCAGGGAC NTCNTC AGCAAT NTCAACG TA NTCN T C CTGGAACTAAAGGGATC IL-2_affmat_ass10CATCAGCATATTCACACATGAATGTTGTTTCAGATCCCTTTAGTTCCAG IL-2_affmat_ass11ATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACA IL-2_affmat_ass12AGATGATGCTTTGACAAAAGGTAATCCATCTGTTCAGAAATTCTACAAT IL-2_affmat_ass13TTTTGTCAAAGCATCATCTCAACACTAACTGGATCCTCTGGTGGCThe site-directed PCR was amplified with the following oligos:

PCR Amplification Oligos (Including 50 bp Homology)

IL-2_site2_assFor 5′-AGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGCACC TACTTCAAGTTCTAC-3′ IL-2_site2_assRev:5′-ACACTGTTGTTATCAGATCTCGAGCAAGTCTTCTTCGGAGATAAGCTTTTGTTCGCCACCA GAGGATCC-3′

The PCT yielded 40 μg of DNA, which was mixed with double digestedpCT302 and electrocompetent EBY100 yeast and electroporated as with thefirst generation library.

Selection of second generation IL-2 library: The library was subjectedto five rounds of selection against IL-2Rβ (FIG. 2B). This selectionmethod was performed exactly as with the first generation library, onlywith modifications to the concentrations of IL-2Rβ used (round 1-1 μM,round 2-100 nM, round 3-30 nM, round 4-30 nM, round 5-10 nM, allmonomeric IL-2Rβ). Upon conclusion of selections, round four and roundfive yeast cultures were spread on SD-CAA plates, which yieldedindividual yeast colonies. 48 individual yeast clones from both roundswere grown in 96-well block format and screened by labeling with 5 nMIL-2Rβ and then SAV-PE. The screen yielded seven high affinity bindersto IL-2Rβ. Amino acid differences among these seven high affinitybinders relative to the corresponding residue in wildtype IL-2 is shownin Table 2 along with the binding affinity for IL-2Rβ.

TABLE 2 K_(d) residue # 74 80 81 85 86 89 92 93 (nM) wt IL-2 Q L R L I II V 280 B1 N F D V V V F 1.6 C5 N V T V V F 10 D10 H F D V V F 1.2 E10 SF D V V F 1.3 G8 N F D V V F 1.5 H4 S T V F 14 H9 F D V V F 1.3CONSENSUS F D V V F

Example 3: IL-2 Mutein Protein Expression and Purification

Human IL-2 variants (amino acids 1-133), the IL-2Rβ ectodomain (aminoacids 1-214), and γ_(c) (amino acids 34-232) were cloned into thepAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67signal sequence and C-terminal hexahistidine tag and produced using thebaculovirus expression system. Baculovirus stocks were prepared bytransfection and amplification in Spodoptera frugiperda (Sf9) cellsgrown in SF900II media (Invitrogen), and protein expression was carriedout in suspension Trichoplusia ni (High Five™) cells grown inBioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed andcaptured from High Five™ supernatants after 48-60 hr by nickel agarose(QIAGEN), concentrated and purified by size exclusion chromatography ona Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assayswere expressed fully glycoslylated. For biotinylated receptorexpression, IL-2Rβ and γ_(c) were cloned into the pAcGP67-A vector witha C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE andhexahistidine tag. Receptor proteins were coexpressed with BirA ligasewith excess biotin (100 uM).

Example 4: Stimulation of CD25⁻ and CD25⁺ Natural Killer (YT-1) Cells

YT-1 and CD25+ YT-1 cells were cultured in RPMI 1640 medium supplementedwith 10% fetal bovine serum, 2 mM L-glutamine, minimum non-essentialamino acids, sodium pyruvate, 25 mM HEPES, and penicillin-streptomycin(Gibco). CD25⁺YT-1 cells were purified as follows: 1×10⁷ cells werewashed with FACS buffer (phosphate buffered saline+2% bovine serumalbumin) and stained with PE-conjugated anti-human CD25 (1:20;Biolegend, San Diego, Calif.) in 1 mL FACS buffer for 20 minutes at 4°C. The stained cells were labeled with paramagnetic microbeads coupledto anti-PE IgG and separated with an LS MACS® separation columnaccording to the manufacturer's instructions (Miltenyi Biotec, BergischGladbach, Germany). Eluted cells were re-suspended in complete RPMImedium at a concentration of 1×10⁵ cells and expanded for subsequentexperiments. Enrichment of cells was monitored via flow cytometry withthe FL-2 channel using an Accuri® C6 flow cytometer.

The dose-response relationships of H9, D10, and 6-6 on YT-1 cells wasdetermined by assaying STATS phosphorylation with flow cytometry (FIGS.4a and 4b ). CD25⁺ or CD25⁻YT-1 cells were washed with FACS buffer andre-suspended in 200 μL FACS buffer with the indicated concentration ofwild-type, 6-6, H9, or D10 in a 96 well plate. Cells were stimulated for20 minutes at room temperature and then fixed by addition offormaldehyde to 1.5% and incubated for 10 min. Cells were permeabilizedwith 100% ice-cold methanol for 20 min on ice, followed by incubation at−80° C. overnight. Fixed, permeabilized cells were washed with excessFACS buffer and incubated with 50 μL Alexa647 conjugated anti-STAT5pY694 (BD Biosciences, San Jose, Calif.) diluted 1:20 in FACS buffer for20 minutes. Cells were washed twice in FACS buffer and mean cellfluorescence determined using the FL-4 channel of an Accuri® C6 flowcytometer.

The CD25 independence of the IL-2 muteins (so called “super-2”molecules) was further tested by taking advantage of awell-characterized mutation of IL-2, phenylalanine to alanine atposition 42 (F42A), which abolishes binding to CD25, yet does not effectits ability to bind to the IL-2Rβ or the IL-2Rγ (Mott, 1995). Thismutation was also introduced into the H9 mutein, yielding H9 F42A. Acomparison of STAT induction by IL-2, IL-2 F42A, H9 and H9 F42A on CD25−and CD25+ YT-1 cells was performed (FIG. 5). While IL-2 F42A mutationright shifted the dose response curve of wild-type IL-2 on CD25+ cellsby approximately 1 log, the F42A mutation had no observable effect onSTAT induction on CD25− cells (FIG. 5a ). In contrast, the dose responsecurves of H9 and H9 F42A were essentially overlapping on both CD25− andCD25+ cells. Thus, these experiments demonstrate that while the IL-2muteins do not apparently benefit from the presence of CD25, theiractivity is insensitive to mutations that disrupt the CD25 interface.

Example 5: Stimulation of CD25− and CD25+ T cells

Human and mouse CD4 T cells were prepared from PBMC (Stanford BloodBank) and spleens and lymph nodes of BALB/C mice, respectively usingantibody-coated CD4 T cell isolation magnetic beads (Stem CellTechnologies and Miltenyi Biotec). For naive cell stimulation assays,cells were used immediately. For generation of in vitro ‘experienced’ Tcells, wells were pre-coated with secondary antibody (Vector Labs) inbicarbonate buffer, pH 9.6 prior to coating plates with anti-CD3 (OKT3for human, 2C11 for mouse, eBiosciences) at 100 ng/mL. T cells wereseeded at 0.1×10⁶ cells/well with soluble anti-CD28 (CD28.2 for human,37.51 for mouse, eBiosciences). Cell were cultured for 3 days with fullTCR stimulated, followed by 2 days rest in conditioned media and 2 daysrest in fresh culture media. Prior to use, live cells were collected byLympholyte-M (Cederlane) centrifugation and counted.

The activity of IL-2 muteins on T cells that were either deficient inCD25 expression or expressed CD25 was assessed (FIG. 6). The doseresponse relationship of wild-type IL-2 and six IL-2 muteins wereassayed for STAT5 phosphorylation at a protein concentration range of 1ng/ml to 1000 ng/ml. The ability of the IL-2 muteins to stimulate STAT5phosphorylation in CD25 deficient T cells correlated well with theiraffinity for the IL-2Rβ. The increase in STAT5 phosphorylation by theIL-2 muteins was two orders of magnitude greater that IL-2.

The ability of IL-2 muteins to stimulate STAT5 phosphorylation onexperienced human CD4+ T cells, which express large amounts of the fullIL-2 receptor complex, CD25 (IL-2Rα), IL-2Rβ, and γ_(c) was alsoassessed (FIG. 7). Human CD4 T cells were in vitro TCR stimulated andrested to generate ‘experienced’ human CD4+CD25+ T lymphocytes. At 1ng/mL, almost no difference in STAT5 phosphorylation was observed. EachIL-2 variant, including wild-type, stimulated over 90% of the cells. At0.1 ng/mL, small differences were observed. Wildtype IL-2 resulted in48% pSTAT5 stimulation, and the IL-2 muteins yielded between 65-79%pSTAT5 stimulation. Therefore, the IL-2 muteins apparently stimulateexperienced human T cells better than wildtype IL-2 but the enhancementis not as pronounced as on cells lacking CD25

Example 6: NK Cell Cytotoxicity Assay

The effect of the D10 IL-2 mutein on Natural Killer cell function,specifically spontaneous and antibody-dependent cell-mediatedcytotoxicity (ADCC) using an EGFR (endothelial growth factorreceptor)-expressing squamous tumor cell line (SCC6) and the EGFRmonoclonal antibody, cetuximab was assessed. Human EGFR-positivesquamous cell carcinoma cell line, SCC6, was obtained as a gift from theJ. Sunwoo Laboratory (Stanford, CA). SCC6 cell line was cultured inDMEM/F12 medium (Invitrogen Life Technologies) supplemented with 10%heat-inactivated FCS (HyClone Laboratories), 100 U/mL penicillin and 100μg/mL streptomycin (both from Invitrogen Life Technologies). Cells weregrown adherent in culture at 37° C. in 5% CO₂. Cetuximab (mouse chimericIgG1 anti-human epidermal growth factor receptor-EGFR; IMC-C225;Erbitux®) was obtained from Bristol-Myers Squibb.

Chromium release was performed as follows: NK cells were isolated from ahealthy donor leukocyte-reduced system (LRS) product containingapproximately 1×10⁹ cells. NK cells were isolated by negative magneticcell sorting using NK cell isolation beads (Miltenyi Biotec) accordingto manufacturer's instructions. NK cells were assessed for purity (>90%purity as defined by CD3⁻CD56⁺ flow cytometry). SCC6 target cells werelabeled with 150 μCi 51_(Cr) per 1×10⁶ cells for 2 h. Percent lysis wasdetermined after 5 h cultures of purified NK cells at variableeffector:target cell ratios of 0:1, 1:1, and 5:1 with 51_(Cr)-labeledSCC6 cells in media alone, cetuximab (100 pg/mL), IL-2 (1000 IU/mL),IL-2 D10 (1 pg/mL), IL-2 D10 (10 pg/mL), or combinations includingcetuximab (100 pg/mL) plus IL-2(1000 IU/mL), cetuximab (100 pg/mL) plusIL-2 D10 (1 pg/mL), or cetuximab (100 pg/mL) plus IL-2 D10 (10 pg/mL).Assay was performed in triplicate. Purified NK cells were cultured with51_(Cr) labelled-SCC6 cells in the presence or absence of cetuximab,IL-2 or IL-2 D10 at variable concentrations. D10 stimulation of NK cellspontaneous cytotoxicity was superior to high-dose IL-2 (FIG. 8,*p=0.008, **p=0.001) with minimal spontaneous cytotoxicity without IL-2or D10 stimulation. ADCC of cetuximab-bound SCC6 was similarly increasedby D10 stimulation compared to high-dose IL-2 or cetuximab alone(*p=0.0005, **p=0.0001). Notably, superior functional enhancement ofcytotoxicity, both spontaneous and ADCC, occurred at all effector:targetratios including 1:1 with D10 compared to high-dose IL-2.

Example 7: IL-2 Muteins Result in Enhanced Memory Phenotype Expansionwith Relatively Low Stimulation of Suppressor-type T Cells (Tregs)

The potency of the IL-2 mutein H9 on the expansion of memory phenotypeCD8⁺ T cells expressing low levels of CD25 but high levels of IL-2Rβγwas assessed in vivo. C57B1/6 mice received either PBS, 20 μg IL-2, 20μg H9, or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes and totalcell counts of splenic CD3⁺CD4⁺CD44^(high) memory phenotype T cells wereassessed by flow cytometry. Splenic cell suspensions were prepared andstained with fluorochrome-conjugated monoclonal antibodies CD3 (clone145-2C11, eBioscience), CD4 (clone RM4-5, Caltag Laboratories), CD8a(clone 53-6.7, BD Biosciences), CD25 (clone PC61, BD Biosciences), CD44(clone IMT, eBioscience) NK1.1 (clone PK136, BD Biosciences) and Thy1.1(clone HIS51, eBioscience). At least 100,000 viable cells were acquiredusing a BD FACSCanto™ II flow cytometer and analyzed using FlowJosoftware (TriStar, Inc.). As shown in FIG. 10(A), treatment with thedisclosed IL-2 mutein resulted in greater expansion of memory phenotypeT cells relative to other treatment modalities with limited expansion ofCD3⁺ CD4⁺CD25^(high)T cells regulatory T cells FIG. 10(B).

Example 8: Reduced In Vivo Toxicity of IL-2 Muteins

It is known that IL-2 treatment can lead to severe adverse effects, suchas acute pulmonary edema, which is currently a limitation preventingmore effective use of IL-2. Accordingly, the toxicity of the disclosedIL-2 muteins relative to IL-2 was assessed (FIG. 11A). C57B1/6 micereceived daily intraperitoneal injections of PBS, 20 μg IL-2, 20 μg H9,or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes for 5 consecutivedays. 6 days after adoptive cell transfer, the lungs were removed andweighed before and after drying overnight at 58° C. under vacuum.Pulmonary wet weight was calculated by subtracting initial pulmonaryweight from lung weight after dehydration.

Example 9: Increased Anti-tumor Activity of IL-2 Muteins In Vivo

The potency of the disclosed IL-2 muteins against tumor cells was testedin vivo. 10⁶ B16F10 melanoma cells in 100 μl RPMI were injected into theupper dermis in the back of mice (3-4 mice per group). Treatmentconsisted of five daily injections of either PBS, 20 μg IL-2, 20 H9, or1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes (IL-2/mAb) and wasstarted one day after tumor nodules were clearly visible and palpable ata size of ˜15mm². The disclosed IL-2 mutein resulted in enhancedanti-tumor activity in vivo as demonstrated in FIG. 11(B).

Example 10: Structural Comparison of IL-2 Muteins and IL-2

Several of the IL-2 muteins were recombinantly expressed in order tomeasure their binding affinity and kinetics for IL-2Rβ by surfaceplasmon resonance (SPR). The affinity between IL-2 and IL-2Rβ wasK_(D)=280 nM. The IL-2 muteins clustered into low, medium, and highaffinity classes. The low affinity IL-2 muteins (5-2 and 6-6) boundIL-2Rβ with K_(D) between 50 and 70 nM, respectively, an affinity gainof 4-6 fold from wild-type IL-2 almost entirely through the L85Vsubstitution. The medium and high affinity mutants selected from thesecondary, site-directed library had K_(D)'s of 10-15 nM (C5, H4), and1.2-1.7 nM (B1, D10, E10, G8, H9), respectively. The affinity increaseswere uniformly manifested in reductions in off-rate, and the highaffinity IL-2 muteins contained a consensus sequence in the randomizedpositions of L80F/R81D/L85V/I86V/I92F.

To understand the structural consequences of the IL-2 muteins, the D10mutein as well as the ternary complex of D10 bound to IL-2Rβ and γ_(c)were crystallized. In the structure of D10 alone, five of the sixmutations are clustered on the B-C loop and within the C helix core, inpositions that do not contact IL-2Rβ. Notably, the B-C helix linkerregion is well-ordered in the electron density map (FIG. 9), compared toother IL-2 structures where this region is either partially orcompletely disordered. Collectively, the F80, V85, and V86 substitutionsappear to collapse into a hydrophobic cluster that stabilizes the loopand ‘pins’ the C-helix into the core of the molecule, packing againsthelix B. The H74 and D81 mutations are solvent exposed and thus, theirstructural roles are less clear, however Asp is a well-known helixN-capping residue that could further contribute the helix C structure.Only one of the six consensus mutations, I92F, was at a position thatcontacts IL-2Rβ in the receptor complex. Phe92 is deeply insertedbetween the C and A helices, contributing only an additional 10 Å² ofmolecular surface buried by IL-2Rβ in the complex compared to Ile92.Thus, its IL-2Rβ contact likely makes only a small contribution to theoverall ˜300-fold affinity gain of D10.

A low-resolution (3.8 Å) structure of the D10 ternary receptor complexwas also determined to assess whether the mutations have perturbed theIL-2Rβ/γc receptor docking geometry. A stable ternary complex of D10 andIL-2Rβ, was crystallized and purified in the absence of CD25. Theoverall IL-2Rβ/γc heterodimeric architecture and mode of cytokine/IL-2Rβcontact in the D10 ternary complex is essentially identical to thepreviously reported quaternary assembly. Thus, the potency increase ofsuper-2 is not due to a structural change in receptor dimerarchitecture, but is likely due to the affinity enhancement.

As discussed earlier, the C-helix of IL-2 appears to undergo subtlerepositioining upon binding to IL-2Rα, as seen in both the binary andquaternary complexes. In contrast, inspection of three wild-typeunliganded structures in the PDB database reveals variability in theC-helix position, consistent with higher B-factors in this helixrelative to the rest of the molecule. Comparison of the structure of D10to that of an unliganded IL-2, and IL-2 in the receptor complexes wasundertaken. It was observed that the C-helix in D10 is more similar tothat seen in the two receptor-bound conformations of IL-2 than the freeforms, having undergone a shift up and into the helical core.

Molecular dynamics (MD) simulations were used to interrogate themechanism by which an IL-2 mutein is endowed with higher bindingaffinity for IL-2Rβ. An atomically detailed Markov state model (MSM) wasconstructed in order to directly probe the relative conformationalflexibility of IL-2 versus IL-2 muteins. The states in this MSM comefrom kinetic clustering of rapidly inter-converting conformationsresulting from atomistic simulations. Each of these metastable statescorresponds to a local minimum in the underlying free energy landscapethat ultimately determines the systems' structure and dynamics. Analysisof the MSM demonstrates that an IL-2 muetin can be more stable thanIL-2, and that IL-2 visits nearly twice as many clusters as an IL-2mutein. For example, IL-2 muteins most populated state has anequilibrium probability of ˜0.20, compared to ˜0.05 for IL-2. Helix B,the B-C loop, and helix C are rigidified in the IL-2 mutein compared toIL-2. As the evolved mutations reside on the B-C loop (H74, D81), andwithin the B and C helix packing interface (F80, V85, V86), bothhelices—not just helix C—benefit from the mutations and undergo acollective stabilization. F92 appears to act as a molecular wedgebetween helix C and helix A, acting as an additional stabilizinginfluence at the more C-terminal end of the helix. That the MDsimulations implicate helix B as also undergoing stabilization insuper-2 was a surprise, since this was not evident from comparison ofIL-2 crystal structures. IL-2Rα binds to IL-2 primarily on the B helixand part of the D helix. The MD simulations suggest the possibility thatbinding of IL-2Rα to IL-2 may rigidify helix B, and this structuralstabilization may be propagated to the B-C loop and helix C. Similar, inprinciple, to the apparent effect of the evolved mutations in the IL-2mutein.

Visualization of the most highly populated conformations from thesimulations for each protein shows that helix C, is far more flexible inIL-2 than the IL-2 mutein, and also that the mutations in the IL-2mutein do indeed stabilize a receptor-bound-like conformation.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. For example, although IL-2 is referred to throughoutthe specification, one of skill in the art would appreciate that themethods and compositions described herein are equally applicable toother cytokines, for example, granulocyte-macrophage colony-stimulatingfactor (GM-CSF), IL-2, IL-3, IL-5, IL-6, or IL-15 with this property.Thus, the invention also includes mutants of GM-CSF, IL-2, IL-3, IL-5,IL-6, and IL-15 with increased binding affinity for their respectivereceptors, as compared to wild-type, and methods for identifying andusing those mutants.

1-16. (canceled)
 17. A nucleic acid encoding an IL-2Rβ binding protein,wherein the equilibrium dissociation constant for IL-2Rβ of the IL-2Rβbinding protein is less than that of wild-type human IL-2 (hIL-2), andwherein said IL-2Rβ binding protein is an IL-2 mutein comprising aminoacid substitutions at positions L80F, R81D, L85V, I86V, and I92Fnumbered in accordance with wild-type hIL-2.
 18. The nucleic acid ofclaim 17, wherein the binding protein further comprises the followingamino acid substitution: F42A.
 19. The nucleic acid of claim 18, whereinthe binding protein further comprises one or more amino acidsubstitutions at positions selected from the group consisting of I24V,P65H, Q74R, Q74H, Q74N, Q74S, I89V, and V931.
 20. The nucleic acid ofclaim 19, wherein position 74 is arginine.
 21. The nucleic acid of claim17, wherein the binding protein has the following substitutions: Q74N,L80F, R81D, L85V, I86V, I89V, and I92F.
 22. The nucleic acid of claim17, wherein the binding protein has the following substitutions: L80F,R81D, L85V, I86V, I89V, I92F, and V931.
 23. The nucleic acid of claim17, wherein the binding protein has the following substitutions: Q74S,L80F, R81D, L85V, I86V, and I92F.
 24. The nucleic acid of claim 17,wherein the binding protein has the following substitutions: Q74H, L80F,R81D, L85V, I86V, and I92F.
 25. The nucleic acid of claim 17, whereinthe binding protein inhibits the interaction between the IL-2Rβ and theIL-2Rγ.
 26. The nucleic acid of claim 25, wherein the binding proteinfurther comprises one or more amino acid substitutions at positions 18,22, 24, 65, 74, 89, 93, and 126 numbered in accordance with wild-typehIL-2.
 27. The nucleic acid of claim 26, wherein the binding protein hasthe following substitutions: L18R, Q22E, L80F, R81D, L85V, I86V, I89V,I92F, V931, and Q126T.
 28. The nucleic acid of claim 26, wherein thebinding protein has the following substitutions: L18R, Q22E, Q74S, L80F,R81D, L85V, I86V, I89V, I92F, V931, and Q126T.
 29. The nucleic acid ofclaim 17, wherein the binding protein has the following substitutions:L18R, Q22E, L80F, R81D, L85V, I86V, I89V, I92F, V931, and Q126T.
 30. Thenucleic acid of claim 17, wherein the binding protein has the followingsubstitutions: Q74N, L80F, R81D, L85V, I86V, and I92F.
 31. The nucleicacid of claim 17, wherein the binding protein further comprises one ormore amino acid substitutions at positions selected from the groupconsisting of 124, F42, K43, P65, Q74, 189, and V93.
 32. The nucleicacid of claim 17, wherein the binding protein further comprises one ormore amino acid substitutions at positions selected from the groupconsisting of F42 and K43.
 33. The nucleic acid of claim 17, wherein thebinding protein further comprises the following amino acid substitution:K43N.
 34. The nucleic acid of claim 17, wherein the binding proteincomprises the following substitutions: F42A, L80F, R81D, L85V, I86V,I89V, and I92F.
 35. The nucleic acid of claim 17, wherein the bindingprotein comprises the following amino acid substitution: Q74N.
 36. Thenucleic acid of claim 32, wherein the binding protein exhibits reducedbinding to an IL-2Rα relative to wild-type IL-2.
 37. The nucleic acid ofclaim 32, wherein the binding protein comprises the followingsubstitutions: L18R, Q22E, L80F, R81D, L85V, I86V, I89V, I92F, V931, andQ126T.
 38. The nucleic acid of claim 37, wherein the binding proteinexhibits reduced binding to IL-2Rγ relative to wild-type IL-2.
 39. Thenucleic acid of claim 37, wherein the binding protein exhibits reducedsignaling through IL-2Rγ relative to wild-type IL-2.
 40. The nucleicacid of claim 17, wherein the binding protein is fused to a heterologouspolypeptide.
 41. The fusion protein of claim 40, wherein theheterologous polypeptide is an antibody Fc region and/or human serumalbumin.
 42. A vector comprising the nucleic acid sequence of claim 17.43. The nucleic acid of claim 17, wherein the IL-2 mutein comprises theamino acid sequence of SEQ ID NO: 12.